Campylobacter jejuni is a leading cause of gastroenteritis in humans in the United States and in other countries throughout the world. According to the Centers for Disease Control, C. jejuni competes with Salmonella species as the leading cause of bacterial gastroenteritis in the United States. In contrast, C. jejuni is a common commensal organism of the gastrointestinal tracts of wild and agriculturally-important animals, which contributes to the large amount of C. jejuni found in the human food supply leading to sporadic cases of disease. Flagellar motility is the only proven virulence and colonization factor of C. jejuni, required to promote infection of humans and avian species for the development of disease or commensalism. C. jejuni produces a single flagellum at one or both poles of the bacterium. Thus, C. jejuni belongs to a significant group of bacterial pathogens, such as Vibrio, Pseudomonas, and Helicobacter species, that are programmed to produce a limited number of flagella and place these organelles only at the poles, unlike more commonly studied peritrichous bacteria such as E. coli and Salmonella species. We have used C. jejuni as a model system to understand regulation of flagellar gene expression and biosynthesis in polarly-flagellated bacterial pathogens. This regulatory system requires the flagellar export apparatus, the FlgSR two-component system and the FlhF GTPase for expression C54-dependent flagellar genes. In addition, FlhF and the putative ATP- binding protein, FlhG, are required for proper flagellar placement, number, or biosynthesis with FlhG possessing an additional function in septation. The objectives of this proposal are to analyze signaling processes in C. jejuni that mediate proper expression of flagellar genes while using the flagellar regulatory system to promote a deeper understanding into the cellular biology of signaling networks, complex organelle development, and bacterial septation. In Aim 1, we will analyze a novel signaling mechanism occurring between the flagellar export apparatus and the FlgS sensor kinase that leads to activation of the FlgSR system. In Aim 2, we will analyze the biology of FlgR, an NtrC-like protein, which contains a unique C- terminus that allows for a likely alternative mechanism of signal transduction and transcriptional initiation. In Aim 3, we will analyze how FlhF influences flagellar gene expression and biosynthesis and the dual functions of FlhG in controlling polar flagellar number and septation. Accomplishment of these aims will aid in understanding: 1) a unique molecular mechanism of signaling between protein systems in bacteria;2) alternative mechanisms of transcriptional initiation for an NtrC-like protein;3) how signaling networks in bacteria are insulated from each other to mediate specificity of signaling;4) complex organelle development;and 5) aspects of bacterial septation. PUBLIC HEALTH RELEVANCE: Campylobacter jejuni produces a flagellum that is required for motility, infection of humans to promote diarrheal disease, and infection of animals to promote colonization. The proposed research will examine how regulatory proteins of C. jejuni function together to control production of flagella and influence other aspects of cellular biology such as bacterial division. By accomplishing research proposed in this work, we may reveal new steps in the production of virulence and colonization factors required by bacteria to promote infection that could be targeted for inactivation by therapeutics.